Secondary Liquefaction in Ethanol Production

ABSTRACT

The invention relates to a method of producing ethanol by fermentation, said method comprising a secondary liquefaction step in the presence of a thermostable acid alpha-amylase or a thermostable maltogenic acid alpha-amylase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/416,393 filed May 9, 2003, which is a 35 U.S.C. 371 nationalapplication of PCT/DK01/00737 filed Nov. 9, 2001, which claims priorityor the benefit under 35 U.S.C. 119 of Danish application nos. PA 200001676 and PA 2000 01854 filed Nov. 10, 2000 and Dec. 11, 2000,respectively, and U.S. provisional application Nos. 60/252,213 and60/256,015 filed Nov. 21, 2000 and Dec. 15, 2000, respectively, thecontents of which are fully incorporated herein by reference,

FIELD OF THE INVENTION

The invention relates to a process for producing ethanol.

BACKGROUND OF THE INVENTION

Ethanol has widespread application as an industrial chemical, gasolineadditive or straight liquid fuel. As a fuel or fuel additive, ethanoldramatically reduces air emissions while improving engine performance.As a renewable fuel, ethanol reduces national dependence on finite andlargely foreign fossil fuel sources while decreasing the netaccumulation of carbon dioxide in the atmosphere. Fermentation processesare used for the production of ethanol. There are a large number ofdisclosures concerning production of alcohol by fermentation, amongwhich are, e.g., U.S. Pat. No. 5,231,017 and CA 1,143,677. EP 138428mentions an Aspergillus niger alpha-amylase preparation for use inliquefaction in the alcohol industry.

There is a need for further improvement of ethanol manufacturingprocesses.

SUMMARY OF THE INVENTION

The invention relates to a method of producing ethanol by fermentation,said method comprising a secondary liquefaction step in the presence ofa thermostable acid alpha-amylase or a thermostable maltogenic acidalpha-amylase. In particular, is provided an improved method forproduction of ethanol based on whole grain as the starch containingstarting material.

Thus, the invention relates to a method of producing ethanol from astarch containing material, preferably based on whole grain, said methodcomprising the steps of: (a) liquefaction of a starch containingmaterial in the presence of an alpha-amylase; (b) jet cooking, (c)liquefaction in the presence of a thermostable acid alpha-amylase or athermostable maltogenic acid alpha-amylase, and (d) saccharification andfermentation to produce ethanol, wherein steps (a), (b), (c) and (d) areperformed in the order (a), (b), (c), (d).

The process of the invention may also comprise one or more additionalsteps, before, in between and/or after step (a), (b), (c) and (d), suchas, e.g, recovering of the ethanol after step (d).

The invention also relates to products obtained or obtainable by theprocesses of the invention and to the use of such products, e.,g., asfuel alcohol or an additive.

The invention in further aspects relates to use of a thermostable acidalpha-amylase or a thermostable maltogenic acid alpha-amylase in asecondary liquefaction step in a process for production of ethanol,particularly from whole grain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a process flow diagram for the preparation of ethanolin accordance with one embodiment of the invention. The primaryliquefaction step may be performed by the presence of the enzymealpha-amylase in the slurry tank while the secondary liquefaction stepis termed “liquefaction” on the diagram.

DETAILED DESCRIPTION OF THE INVENTION

Ethanol Production

The present invention provides a process of producing ethanol, inparticular improvement of the secondary liquefaction step in a processof producing ethanol from dry milled whole grain.

The invention provides a method of producing ethanol by fermentation,said method comprising a secondary liquefaction step in the presence ofa thermostabte acid alpha-amylase or a thermostabte maltogenic acidalpha-amylase. A particularly interesting embodiment relates to afermentation process of the invention where the starting material iswhole grain which has been partitioned into finer parts, preferably bydry milling.

Thus, the invention in one aspect relates to a method of producingethanol from a starch containing material, said method comprising thesteps of:

a) liquefaction of a starch containing material in the presence of analpha-amylase;

b) jet cooking,

c) liquefaction in the presence of a thermostable acid alpha-amylase;

d) saccharification and fermentation to produce ethanol;

wherein steps (a), (b), (c) and (d) are performed in the order (a), (b),(c), (d).

Raw Material

In one embodiment, the starch containing material is selected from thegroup consisting of: tubers roots and whole grain; and any combinationsof the forgoing. In one embodiment, the starch containing material isobtained from cereals. The starch containing material may, e.g., beselected from the groups consisting of corns, cobs, wheat, barley,cassava, sorghum, rye, milo and potatoes; or any combination of theforgoing.

In the ethanol processes of the invention, the starting raw material ispreferably whole grain or at least mainly whole grain. A wide variety ofstarch containing whole grain crops may be used as raw materialincluding: corn (maize), milo, potato, cassava, sorghum, wheat, andbarley.

Thus, in one embodiment, the starch containing material is whole grainselected from the group consisting of corn (maize), milo, potato,cassava, sorghum, wheat, and barley; or any combinations thereof. In apreferred embodiment, the starch containing material is whole grainselected from the group consisting of corn, wheat and barley or anycombinations thereof.

The raw material may also consist of or comprise a side stream fromstarch processing—e.g., C6 carbohydrate containing process streams thatare not suited for production of syrups. In other embodiments, the rawmaterial does not consist of or comprise a side stream from starchprocessing.

Process Steps

The main process steps of the present invention may in one embodiment bedescribed as separated into the following main process stages: milling(when whole grain is used as raw material), primary liquefaction,heat-treatment as provided by jet-cooking, secondary liquefaction,saccharification, fermentation, distillation.

In a preferred embodiment, the method of the invention comprises priorto step (a) the steps of: i) dry milling of whole grain; and ii) forminga slurry comprising the milled grain and water.

The individual process steps of alcohol production may be performedbatch wise or as a continuous flow. For the invention processes whereall process steps are performed batch wise, or processes where allprocess steps are performed as a continuous flow, or processes where oneor more process step(s) is(are) performed batch wise and one or moreprocess step(s) is(are) performed as a continuous flow, are equallycontemplated.

The cascade process is an example of a process where one or more processstep(s) is(are) performed as a continuous flow and as such contemplatedfor the invention. For further information on the cascade process andother ethanol processes consult The Alcohol Textbook. Ethanol productionby fermentation and distillation. Eds. T. P. Lyons, D. R. Kesall and J.E. Murtagh. Nottingham University Press, 1995.

Milling

Thus, in a preferred embodiment of the process of the invention, thestarch containing material is whole grain and the method comprises astep of milling the whole grain before step (a), i.e., before theprimary liquefaction. In other words, the invention also encompassesprocesses of the invention, wherein the starch containing material isobtainable by a process comprising milling of whole grain, preferablydry milling, e.g., by hammer or roller mils. Grinding is also understoodas milling.

In particular embodiments, the process of the invention furthercomprises prior to the primary liquefaction step (i.e., prior to step(a), the steps of:

i. milling of whole grain.

ii. forming a slurry comprising the milled grain and water to obtain thestarch containing material.

The whole grain is milled in order to open up the structure and allowingfor further processing. Two processes of milling are normally used inalcohol production; wet and dry milling. The term “dry milling” denotesmilling of the whole grain. In dry milling the whole kernel is milledand used in the remaining part of the process. Wet milling gives a goodseparation of germ and meal (starch granules and protein) and is with afew exceptions applied at locations where there is a parallel productionof syrups.

Thus, in a preferred embodiment of the invention, dry milling is usedsince the secondary liquefaction step is advantageously included in drymilling processes for producing ethanol.

Liquefaction

In the liquefaction process the starch containing material, preferablyin the form of milled whole grain raw material, is broken down(hydrolyzed) into maltodextrins (dextrins). In a preferred embodiment,in the primary liquefaction process of the invention the starchcontaining material, preferably in the form of milled whole grain rawmaterial, is hydrolyzed to a DE (an abbreviation for dextroseequivalent) higher than 4. DE stands for “Dextrose equivalents” and is ameasure of reducing ends on C6 carbohydrates. Pure glucose has DE of100. Glucose (also called dextrose) is a reducing sugar. Whenever anamylase hydrolyzes a glucose-glucose bond in starch, two new glucoseend-groups are exposed, At least one of these can act as a reducingsugar. Therefore the degree of hydrolysis can be measured as an increasein reducing sugars. The value obtained is compared to a standard curvebased on pure glucose—hence the term dextrose equivalent. The DE may,e.g., be measured using Fehlings liquid by forming a copper complex withthe starch using pure glucose as a reference, which subsequently isquantified through iodometric titration. In other words, DE (dextroseequivalent is defined as the amount of reducing carbohydrate (measuredas dextrose-equivalents) in a sample expressed as w/w % of the totalamount of dissolved dry matter. It may also be measured by theneocuproine assay (Dygert, Li Floridana, 1965, Anal. Biochem. No 368).The principle of the neocuproine assay is that CuSO₄ is added to thesample, Cu²⁺ is reduced by the reducing sugar and the formed neocuproinecomplex is measured at 450 nm.

The hydrolysis may be carried out by acid treatment or enzymatically.The liquefaction is preferably carried out by enzymatic treatment,preferably an alpha-amylase treatment. In one embodiment, theliquefaction is carried out by preparing a slurry comprising milled rawmaterial, preferably milled whole grain, and water, heating the slurryto between 60-95° C., preferably 80-85° C., and the enzyme(s) is (are)added to initiate liquefaction (thinning). This is also termed the“primary liquefaction”, ie., it occurs before the process step ofjet-cooking (step (b)). The liquefaction in the process of the inventionis performed at any conditions ie., e.g., pH, temperature and time)found suitable for the enzyme in question. Within the scope is a methodof the invention, wherein the liquefaction in step (a) is performed at60-95° C. for 10-120 min, preferably at 75-90° C. for 15-40 min. In oneembodiment, the liquefaction in step (a) is performed at a pH in therange of about pH 4-7, preferably pH about 4.5-6.5. The pH of the slurrymay by adjusted or not, depending on the properties of the enzyme(s)used. Thus, in one embodiment the pH is adjusted, e.g., about 1 unitupwards, e.g., by adding NH₃. The adjusting of pH is advantageously doneat the time when the alpha-amylase is added. In a preferred embodiment,the pH is not adjusted and the alpha-amylase has a correspondingsuitable pH-activity profile, such as being active at a pH about 4.

After the primary liquefaction step, the slurry is preferably jet-cookedat appropriate conditions to further gelatinize the starch, such as,e.g., at a temperature between 95-140° C., preferably 105-125° C. toensure the gelanitization. In one embodiment, the jet-cooking in step(b) is performed under conditions 1-10 min, 105-150° C. and e.g., pH4-7; preferably for 1-5 min, 105-120° C. and e.g., pH 4.5-6; such as,e.g., about 5 min, about 105° C., and e.g., pH about 5.0. As usedherein, generally, the term jet-cooking also covers any other methodwhich can be used to obtain a similar result.

Then the slurry is preferably cooled, eg., to about 60-95° C. and moreenzyme(s) is (are) added to obtain the final hydrolysis; the later istermed “secondary liquefaction”, i.e., liquefaction after jet-cookingwhich by the process of the invention is obtained by addition of atleast a thermostable acid alpha-amylase or a thermostable maltogenicacid alpha-amylase.

The secondary liquefaction in step (c) is performed at suitableconditions (pH, temperature and process time). The secondaryliquefaction in step (c) may e.g., be performed at 60-95° C. for 10-120min, preferably at 70-85° C. for 15-80 min and at pH 4.5-6.5. In oneembodiment, the pH is not adjusted for the secondary liquefaction. Inpreferred embodiment, the pH during the secondary liquefaction is atmost about 5.

In one preferred embodiment, in the secondary liquefaction step in themethod of the invention the starch containing material, e.g., obtainedfrom dry milled whole grain, is hydrolyzed to a DE in the range of about5-15, e.g., 8-15, 8-14, such as, such as a DE in the range about 10-14.,e.g., about 10-12.

The liquefaction process (both the primary and the secondaryliquefaction process) is carried out at a suitable pH, e.g., at a pH inthe range 4.5-6.5, such as at a pH between about 5 and about 6.

Milled and liquefied whole grain are also known as mash.

Saccharification

To produce low molecular sugars DP₁₋₂ that can be metabolized by yeast,the maltodextrin from the liquefaction is preferably further hydrolyzed;this is also termed “saccharification”. The hydrolysis may be doneenzymatically by the presence of a glucoamylase. An alpha-glucosidaseand/or an acid alpha-amylase may also be present in addition to theglucoamylase.

A full saccharification step may last up to 72 hours. However, thesaccharification and fermentation (SSF) may be combined, and in someembodiments of the invention a pre-saccharification step of 1-4 hoursmay be included. Pre-saccharification is carried out at any suitableprocess conditions. In a preferred embodiment, the pre-saccharificationis carried out at temperatures from 30-65° C. such as around 60° C., andat, e.g., a pH in the range between 4-5, especially around pH 4.5.

Thus in one embodiment, the method of the invention may further comprisea pre-saccharification step, as described herein, which is performedafter the secondary liquefaction step (c) and before step (d).

In other embodiments, the process of the invention does not comprise apre-saccharification and the saccharification is essentially onlyperformed during fermentation, e.g., by the presence of a glucoamylaseand optionally phytase.

Fermentation

The microorganism used for the fermentation is added to the mash and thefermentation is ongoing until the desired amount of ethanol is produced;this may, e.g., be for 24-96 hours, such as 35-60 hours. The temperatureand pH during fermentation is at a temperature and pH suitable for themicroorganism in question, such as, e.g, in the range about 26-34° C.,e.g., about 32° C., and at a pH, e.g., in the range about pH 3-6, e.g.,about pH 4-5.

In a preferred embodiment, a simultaneous saccharification andfermentation (SSF) process is employed where there is no holding stagefor the saccharification, meaning that yeast and saccharification enzymeis added essentially together. In one embodiment, when doing SSF isintroduced a pre-saccharification step at a temperature above 50° C.,just prior to the fermentation.

In one embodiment, the fermentation is carried out in the presence ofglucoamylase and/or protease.

In a further embodiment, the addition of a thermostable acidalpha-amylase or a thermostable maltogenic acid alpha-amylase in thesecondary liquefaction step in the process of the invention may make itpossible to substitute the presence of glucoamylase activity in thefermentation step. Thus, one embodiment relates to a process of theinvention for the production of ethanol, without addition ofglucoamylase in the fermentation step or prior to the fermentation step.

Distillation

The method of the invention may further comprise recovering of theethanol; hence the alcohol may be separated from the fermented materialand purified. Following the fermentation the mash may be distilled toextract the ethanol. Ethanol with a purity of up to, e.g., about 96 vol.% ethanol can be obtained by the process of the invention.

Thus, in one embodiment, the method of the invention further comprisesthe step of: (e) distillation to obtain the ethanol. The fermentation instep (d) and the distillation in step (e) may be carried outsimultaneously and/or separately/sequentially; optionally followed byone or more process steps for further refinement of the ethanol.

By-Products from Distillation and Recycling:

In one embodiment of the process of the invention, the aqueousby-product (“Whole Stillage”, cf. FIG. 1) from the distillation processis separated into two fractions, for instance by centrifugation: 1) “WetGrain” (solid phase, see FIG. 1), and 2) “Thin Stillage” (Supernatant,see FIG. 1).

In one embodiment, the starch containing material entering the processof the invention is dry milled whole grain, and the method of theinvention comprising steps (a), (b), (c), (d), and (e) further comprisesthe steps of:

(f) separation of Whole Stillage produced by of the distillation in step(e), into wet grain and Thin stillage; and

(g) recycling Thin stillage to the starch containing material prior tothe primary liquefaction of step (a).

In one embodiment, in the process of the invention, the Thin Stillage(cf. FIG. 1) is recycled to the milled whole grain slurry.

The Wet Grain fraction may be dried, typically in a drum dryer. Thedried product is referred to as “Distillers Dried Grains” (see FIG. 1),and can be used, e.g., as animal feed.

The Thin Stillage fraction may be evaporated providing two fractions(see FIG. 1):

(i) a Condensate fraction of 4-6% DS (mainly of starch, proteins, andcell wall components), and

(ii) a Syrup fraction, mainly consisting of limit dextrins and nonfermentable sugars, which may be introduced into a dryer together withthe Wet Grains (from the Whole Stillage separation step) to provide aproduct referred to as “Distillers Dried Grain”, which also can be usedas animal feed.

“Thin Stillage” is the term used for the supernatant of thecentrifugation of the Whole Stillage (see FIG. 1). Typically, the ThinStillage contains 4-6% DS (mainly starch and proteins) and has atemperature of about 60-90° C.

In another embodiment Thin Stillage is not recycled, but the condensatestream of evaporated Thin Stillage is recycled to the slurry containingthe milled whole grain to be jet cooked.

One embodiment of the invention relates to a method of producingethanol, said method comprising the steps of:

a) primary liquefaction of a starch containing material in the presenceof alpha-amylase activity;

b) jet cooking the material of step(a);

c) secondary liquefaction of the material of step (b) in the presence ofa thermostable acid alpha-amylase or a thermostable maltogenic acidalpha-amylase; and

d) saccharification and fermentation to produce ethanol;

wherein steps (a) (b), (c) and (d) are performed in the order (a), (b),(c), (d).

Optionally the saccharification and fermentation may be performed inseparate steps. Thus the invention also relates to a method of producingethanol, said method comprising the steps of:

a) primary liquefaction of a starch containing material in the presenceof alpha-amylase activity,

b) jet cooking the material of step(a);

c) secondary liquefaction of the material of step (b) in the presence ofa thermostable acid alpha-amylase or a thermostable maltogenic acidalpha-amylase, and

d) saccharification;

e) fermentation to produce ethanol;

wherein steps (a), (b), (c) and (d) are performed in the order (a), (b),(c), (d) and wherein (e) is performed simultaneously to or following(d).

The invention in also relates to a method of producing ethanol, saidmethod comprising the steps of:

a) dry milling of whole grain;

b) forming a slurry comprising the milled grain and water;

c) liquefaction in the presence of an alpha-amylase;

d) jet cooking;

e) liquefaction in the presence of a thermostable acid alpha-amylase ora thermostable maltogenic acid alpha-amylase;

f) saccharification in the presence of phytase and/or glucoamylase andfermentation to produce ethanol;

g) distillation;

optionally followed by one or more process steps for further refinementof the ethanol;

wherein steps (a), (b), (c), (d), (e) and (f) are performed in the order(a), (b), (c), (d), (e), (f); and

wherein step (g) is performed simultaneously with step (f) and/or afterstep (f).

In a further embodiment, the method of the invention for producingethanol may comprise the following steps:

a) milling whole grain;

b) making a slurry comprising the milled whole grain and water;

c) liquefying in the presence of an alpha-amylase;

d) saccharifying in the presence of a glucoamylase and fermenting usinga microorganism,

f) distillation of the fermented material, providing two streams 1)alcohol and 2) Whole Stillage;

(g1) recovering alcohol for further refinement; optionally,

(g2) separating the Whole Stillage into two fractions of, 1) Wet Grain,and 2) Thin Stillage;

(h1) the whole grain fraction is dried to provide a protein containingproduct, and optionally

(h2) the Thin Stillage is evaporated providing two streams: 1)condensate stream and 2) syrup;

wherein the Thin Stillage and optionally the condensate from step (h2)is recycled to step (b) with or without further treatment.

Enzyme Activities

Alpha-Amylase

The “primary liquefaction” is preferably performed in the presence of analpha-amylase, e.g., derived from a micro-organism or a plant. Preferredalpha-amylases are of fungal or bacterial origin Bacillus alpha-amylases(often referred to as “Termamyl-like alpha-amylases”), variant andhybrids thereof, are specifically contemplated according to theinvention. Well-known Termamyl-like alpha-amylases include alpha-amylasederived from a strain of B. licheniformis (commercially available asTermamyl™), B. amyloliquefaciens, and B. stearothermophilusalpha-amylase. Other Termamyl-like alpha-amylases include alpha-amylasederived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB12513 or DSM 9375, all of which are described in detail in WO 95/26397,and the alpha-amylase described by Tsukamoto et al., Biochemical andBiophysical Research Communications, 1988, 151: 25-31. In the context ofthe present invention a Termamyl-like alpha-amylase is an alpha-amylaseas defined in WO 99/19467 on page 3, line 18 to page 6, line 27.Contemplated variants and hybrids are described in WO 96/23874, WO97/41213, and WO 99/19467, and include the Bacillus stearothermophilusalpha-amylase (BSG alpha-amylase) variant, alpha-amylase TTC, having thefollowing mutations delta(181-182)+N193F (also denotedI181*+G182*+N193F) compared to the wild-type amino acid sequence setforth in SEQ ID NO: 3 disclosed in WO 99/19467. Contemplatedalpha-amylases derived from a strain of Aspergillus includes Aspergillusoryzae and Aspergillus niger alpha-amylases.

Commercial alpha-amylase products and products containing alpha-amylasesinclude TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ SC and SAN™ SUPER,(Novozymes A/S, Denmark) and DEX-LO™, SPEZYME™ AA, and SPEZYME™ DELTA AA(from Genencor Int.).

Other contemplated alpha-amylases are the KSM-K36 alpha-amylasedisclosed in EP 1,022,334 and deposited as FERM BP 6945, and the KSM-K38alpha-amylases disclosed in EP 1,022,334, and deposited as FERM BP-6946.Also variants thereof are contemplated, in particular the variantsdisclosed in Danish patent application no. PA 2000 11533 (from NovozymesA/S.

The “secondary liquefaction” is performed in the presence of analpha-amylase, in particular a thermostable acid alpha-amylase or athermostable maltogenic acid alpha-amylase as described herein for usein the secondary liquefaction step in the process of the invention. Thealpha-amylase is preferably derived from a micro-organism, includingfungal and bacterial, or derived from a plant. Preferred thermostabteacid alpha-amylases are of bacterial origin. Preferred thermostablemaltogenic acid alpha-amylases are of fungal orgin.

It is understood that enzymes are added in an effective amount for theactual conditions (temperature, pH) of the process, e.g., that thethermostable acid alpha-amylase is added in an amount effective in step(c).

In further embodiments of the process of the invention, in step (c)apart from the addition of the thermostable acid alpha-amylase is alsoadded an alpha-amylase which is not a thermostable acid alpha-amylase.

The term “thermostable” in the context of a thermostable acidalpha-amylase means in one embodiment that the enzyme is active up to90° C. at pH 5.0 using a 0.1 M citrate buffer and 4.3 mM Ca²⁺.

The thermostable acid alpha-amylase should have activity at the pHpresent during the liquefaction and fermentation, such as, e.g., at a pHin the range pH 2.5-5.5 using a 0.1 M citrate buffer and 4.3 mM Ca²⁺.The enzyme should preferably at least be active in the range at pH 3-5.It is understood that the enzyme may also be active outside the pHranges mentioned.

Examples of thermostable acid alpha-amylases as used herein are thealpha-amylases selected from the group consisting of LE399; theAspergillus oryzae TAKA alpha-amylase (EP 238 023); the Aspergillusniger alpha-amylase disclosed in EP 383,779 B2 (section [0037] (see alsothe cloning of the A. niger gene in Example 1); the Aspergillus nigeralpha-amylase disclosed in Example 1 of EP 140,410; Commercial fungalalpha-amylases FUNGAMYL® (Novozymes A/S); and Clarase™ (from GenencorInt., USA), the latter both derived from Aspergillus.

LE399 is a hybrid alpha-amylase. Specifically, LE399 comprises the 445C-terminal amino acid residues of the Bacillus licheniformisalpha-amylase (shown in SEQ to NO: 4 of WO 99/19467) and the 37N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467), withthe following substitution:G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the numberingin SEQ ID NO: 4 of WO 99/19467).

By the expression “secondary liquefaction in the presence of athermostable acid alpha-amylase” is understood as liquefaction in thesecondary liquefaction step in the process of the invention by treatmentwith an effective amount of a thermostable acid alpha-amylase” asdefined herein.

The thermal/pH stability may be tested using, e.g., the followingmethod: 950 micro liter 0.1 M Citrate+4.3 mM Ca²⁺ buffer is incubatedfor 1 hour at 60° C. 50 micro liters enzyme in buffer (4 AFAU/ml) isadded. 2×40 micro liter samples are taken at 0 and 60 minutes andchilled on ice. The activity (AFAU/ml) measured before incubation (0minutes) is used as reference (100%). The decline in percent iscalculated as a function of the incubation time. To determine theThermal stability the test is repeated using different temperatures, forinstance 50, 60, 70, 80 and 90° C. To determine the pH stability thetest is repeated using different pH's, for instance, pH 2.5; 3; 3.5; 4;4.5; 5.

An example of an alpha-amylase, in particular a thermostabte maltogenicacid alpha-amylase, used in the process of the invention is thealpha-amylase having the amino acid sequence set forth in SEQ ID NO: 1(also named SP288) and variants thereof having one or more amino acidresidues which have been deleted, substituted and/or inserted comparedto the amino acid sequence of SEQ ID NO: 1; which variants havealpha-amylase activity, preferably being a thermostable maltogenic acidalpha-amylase.

Thus, the alpha-amylase used in the secondary liquefaction step in theprocess of the invention, may, e.g., be an alpha-amylase, in particulara thermostable maltogenic acid alpha-amylase, having an amino acidsequence which has at least 70% identity to SEQ ID NO: 1 preferably atleast 75%, 80%, 85% or at least 90%, e.g., at least 95%, 97%, 98%, or atleast 99% identity to SEQ ID NO: 1. In the present context, the degreeof identity between two amino acid sequences is described by theparameter “identity” given in %. For purposes of the present invention,the degree of identity between two amino acid sequences is preferablydetermined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153)using the LASERGENE™ MEGALIGN™ software (DNASTAR, inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10, and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, windows=5, and diagonals=5].

Thus, the thermostable maltogenic acid alpha-amylase used in the processof the invention may, e.g., also be an alpha-amylase, in particular athermostable maltogenic acid alpha-amylase, having an amino acidsequence which is a fragment of SEQ ID NO: 1. When using the term“alpha-amylase” or “thermostable maltogenic acid alpha-amylase” in thecontext of variants and fragments of, e.g., SEQ ID NO: 1, it is to beunderstood that the enzyme is capable of being enzymatically active.When used herein, a “fragment” of SEQ ID NO: 1 is a polypeptide havingone or more amino acids deleted from the amino and/or carboxyl terminusof this amino acid sequence. Preferably, a fragment contains at least 50amino acid residues or at least 100 amino acid residues.

The enzyme given by SEQ ID NO: 1 is also disclosed in Boel E. et al.,“Calcium binding in alpha-amylases: an X-ray diffraction study at 2.1-Aresolution of two enzymes from Aspergillus”. Biochemistry, 1980,29:6244-6249, e.g., in table 1 and under “Material and Methods” of thesame.

Other examples of alpha-amylases which may be used in the secondaryliquefaction step in the process of the invention, is the alpha-amylasedisclosed in Agric. Biol. Chem., 1979, 43:1165-1171 by Guy-Jean Moulinand Pierre Galzy.

It is understood that the enzyme are added in an effective amount forthe actual conditions (temperature, pH) of the process, e.g., that thethermostable maltogenic acid alpha-amylase is added in an amounteffective in step (c).

In further embodiments of the process of the invention, in the secondaryliquefaction step (c) apart from the addition of a thermostablemaltogenic acid alpha-amylase (e.g., the alpha-amylase of SEQ ID NO: 1and variants thereof as described herein) is also added an alpha-amylasewhich is not a thermostable maltogenic acid alpha-amylase as definedherein, such as e.g., the alpha-amylase TTC.

The thermostable maltogenic acid alpha-amylase should have activity atthe pH present during the liquefaction and fermentation; such as e.g.,at a pH in the range pH 2.5-5.5 using a 0.1 M citrate buffer and 4.3 mMCa²⁺, a substrate consisting of DE 12 alpha-amylase TTC liquefied cornstarch at 30% dry substance. The enzyme should preferably at least beactive in the range at pH 3-5, preferably at least pH 2.5-5. It isunderstood that the enzyme may also be active outside the pH rangesmentioned.

The term “maltogenic” in the context of the invention, means that theenzyme is capable of releasing a relatively high amount of α-maltose asa product of its enzymatic activity.

In a particular interesting embodiment, the term “maltogenic” means thatthe enzyme using a DE 12 alpha-amylase TTC liquefied corn starch at 30%dry substance at 60° C., pH 4.5 and dosing the enzyme at 1 AFAU/g drysubstance, the enzyme will in 24 hours catalyze the formation of atleast 15%, or at least 20%, at least 25%, at least 30 w/w maltose asbased on the total amount of starch. The maltose content may forinstance be measured by with HPLC as known by the person skilled in theart.

The term “DE 12 alpha-amylase TTC liquefied corn starch” in this contextmeans that the substrate used for testing the maltogenicity of thealpha-amylase enzyme, is corn starch liquefied to a DE of 12 withalpha-amylase TTC.

The term “thermostable” means that the enzyme is relatively stable athigher temperatures. In one embodiment, the enzyme will maintain morethan 90% of its activity for 1 hour at 70° C. using a DE 12alpha-amylase TTC liquefied corn starch at 30% dry substance assubstrate, pH 5.5, 0.1 M citrate buffer and 4.3 mM Ca²⁺.

The term “acid” means that the enzyme is relatively stable at low pH. Inone embodiment, the enzyme will maintain more than 70% of its activityin the range from pH 3.5-5.0 (e.g., at pH 4), or preferably in the rangefrom pH 3.8-4.7 (e.g., at pH 4.2) at the conditions: substrate DE 12alpha-amylase TTC liquefied corn starch at 30% dry substance,temperature of 40° C., 0.1 M citrate buffer and 4.3 mM Ca²⁺.

In one embodiment, the pH window (profile) of the enzyme used in thesecondary liquefaction step in the process of the invention is asfollows: the maximum activity of the enzyme is found at approximately pH4.2 and/or the enzyme will maintain more than 70% of its activity in therange from pH 3.5-5.0 at the conditions; substrate is DE 12alpha-amylase TTC liquefied corn starch at 30% dry substance,temperature of 40° C., 0.1 M citrate buffer and 4.3 mM Ca²⁺.

In one embodiment the temperature window (profile) of the alpha-amylaseenzyme used in the secondary liquefaction step in the process of theinvention is as follows; the enzyme will maintain more than 80% of itsactivity for 15 min in the range from 50-80° C. using a DE 12alpha-amylase TTC liquefied corn starch at 30% dry substance assubstrate, pH 5.5, 0.1 M citrate buffer and 4.3 mM Ca²⁺.

The alpha-amylase enzyme used in the secondary liquefaction step in theprocess of the invention may catalyze the hydrolysis ofbeta-cyclodextrins which is one of the characteristics of the enzymehaving the amino acid sequence of SEQ ID NO: 1.

By the expression “secondary liquefaction in the presence of athermostable maltogenic acid alpha-amylase” is understood liquefactionin the secondary liquefaction step by treatment with an effective amountof a thermostable maltogenic acid alpha-amylase as defined herein. Thealpha-amylase used in the secondary liquefaction is preferably athermostable maltogenic acid alpha-amylase. The term “thermostablemaltogenic acid alpha-amylase”, means that the alpha-amylase is boththermostable, acid and maltogenic as defined herein. In one embodiment,the alpha-amylase is at least thermostable and acid as defined herein,optionally being maltogenic as defined herein.

The thermostable maltogenic acid alpha-amylase may be employed in theprimary liquefaction step; however, the maximum effect is obtained ifthe enzyme is added the secondary liquefaction step.

Enzyme Activities used During Saccharification or SSF

Glucoamylase

The saccharification step or the simultaneous saccharification andfermentation step (SSF) may be carried out in the presence of aglucoamylase. The glucoamylase may be of any origin, e.g., derived froma microorganism or a plant. Preferred is glucoamylase of fungal orbacterial origin selected from the group consisting of Aspergillus nigerglucoamylase, in particular A. niger G1 or G2 glucoamylase (Boel et al.,1984, EMBO J. 3 (5): 1097-1102), or variants thereof, such as disclosedin WO 92/00381 and WO 00/04136: the A. awamori glucoamylase (WO84/02921), A. oryzae (Agric. Biol. Chem., 1991, 55 (4): 941-949), orvariants or fragments thereof.

Other contemplated Aspergillus glucoamylase variants include variants toenhance the thermal stability: G137A and G139A (Chen et al., 1996, Prot.Engng. 9: 499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Engng.8: 575-582); N182 (Chen et al., 1994, Biochem. J. 301: 275-281);disulphide bonds, A246C (Fierobe et al., 1996, Biochemistry, 35:8698-8704; and introduction of Pro residues in position A435 and S436(Li et al., 1997, Protein Engng. 10: 1199-1204. Furthermore, Clark Fordpresented a paper on Oct. 17, 1997, ENZYME ENGINEERING 14, Beijing/ChinaOct. 12-17, 1997, Abstract number: Abstract book p. 0-61. The abstractsuggests mutations in positions G137A, N20C/A27C, and S30P in anAspergillus awamori glucoamylase to improve the thermal stability. Otherglucoamylases include Talaromyces glucoamylases, in particular derivedfrom Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S.Pat. No. Re. 32,153), Talaromyces duponti, Talaromyces thermopiles (U.S.Pat. No. 4,587,215). Bacterial glucoamylases contemplated includeglucoamylases from the genus Clostridium, in particular C.thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO86/01831).

Commercial products include SAN™ SUPER™ and AMG™ E (from Novozymes A/S).

Protease

Addition of protease(s) in the saccharification step, the SSF stepand/or the fermentation step increase(s) the FAN (Free amino nitrogen)level and increase the rate of metabolism of the yeast and further giveshigher fermentation efficiency.

Suitable proteases include microbial proteases, such as fungal andbacterial proteases. Preferred proteases are acidic proteases, i.e.,proteases characterized by the ability to hydrolyze proteins underacidic conditions below pH 7.

In a preferred embodiment, the protease is selected from the group offungal proteases, such as e.g., an acid fungal protease derived from astrain of Aspergillus.

Suitable acid fungal proteases include fungal proteases derived fromAspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra,Irpex, Penicillium, Sclerotiumand Torulopsis. Especially contemplatedare proteases derived from Aspergillus niger (see, e.g., Koaze et al.,1964, Agr. Biol. Chem. Japan, 28: 216), Aspergillus saitoi (see, e.g.,Yoshida, 1954, J. Agr. Chem. Soc. Japan, 28:66), Aspergillus awamori(Hayashida et as., 1977, Agric. Biol. Chem., 42(5): 927-933, Aspergillusaculeatus (WO 95/02044), or Aspergillus oryzae, such as the pepAprotease; and acidic proteases from Mucor pusillus or Mucor miehei.

Also contemplated are neutral or alkaline proteases, such as a proteasederived from a strain of Bacillus. Bacterial proteases, which are notacidic proteases, include the commercially available products Alcalase®and Neutrase® (available from Novozymes A/S.

Additional Enzymes:

One or more additional enzymes may also be used duringsaccharification/pre-saccharification or SSF. Additional enzymes includee.g., pullulanase and/or phytase. Thus, in one embodiment, aglucoamylase and/or phytase is added in order to promote thefermentation.

Phytase:

The phytase used according to the invention may be any enzyme capable ofeffecting the liberation of inorganic phosphate from phytic acid(myo-inositol hexakisphosphate) or from any salt thereof (phytates).Phytases can be classified according to their specificity in the initialhydrolysis step, viz. according to which phosphate-ester group ishydrolyzed first. The phytase to be used in the invention may have anyphytase specificity, e.g., a 3-phytase (EC 3.1.3.8), a 6-phytase (EC3.1.3.26) or a 5-phytase.

A suitable dosage of the phytase is e.g., in the range 5,000-250,000FYT/g DS, particularly 10,000-100,000 FYT/g DS.

The phytase activity may be determined FYT units, one FYT being theamount of enzyme that liberates 1 micromole inorganic ortho-phosphateper min. under the following conditions: pH 5.5, temperature 37° C.;substrate: sodium phytate (C₆H₆CO₂₄P₆Na₁₂) at a concentration of 0.0050mole/I.

The phytase may be of any origin, such as, e.g., microbial, such as, eg., derived from a strain of Peniophra lycii or Aspergillus oryzae. Itmay be produced recombinantly or non-recombinantly. The phytase may bederived e.g., from plants or microorganisms, such as bacteria or fungi,e.g., yeast or filamentous fungi.

The plant phytase may be from wheat-bran, maize, soy bean or lilypollen. Suitable plant phytases are described in Thomlinson et al, 1962,Biochemistry, 1: 166-171; Barrientos et al., 1994, Plant. Physiol., 106:1489-1495; WO 98/05785; WO 98/20139.

A bacterial phytase may be from genus Bacillus, Pseudomonas orEscherichia, specifically the species B. subtilis or E. coli. Suitablebacterial phytases are described in Paver and Jagannathan, 1982, Journalof Bacteriology 151:1102-1108; Cosgrove, 1970, Australian Journal ofBiological Sciences 23:1207-1220; Greiner et al., 1993, Arch. Biochem.Biophys., 303, 107-113: WO 98/06856; WO 97/33976; WO 97/48812.

A yeast phytase or myo-inositol monophosphatase may be derived fromgenus Saccharomyces or Schwanniomyces, specifically speciesSaccharomyces cerevisiae or Schwanniomyces occidentalis. Suitable yeastphytases are described in Nayini et al., 1984, Lebensmittel Wissenschaftund Technologie 17:24-26; Wodzinski et al., Adv. Appl. Microbiol., 42:263-303; AU-A-24840/95;

Phytases from filamentous fungi may be derived from the fungal phylum ofAscomycota (ascomycetes) or the phylum Basidiomycota, e.g., the genusAspergillus, Thermomyces (also called Humicola), Myceliophthora,Manascus, Penicillium, Peniophora, Agrocybe, Paxillus, or Trametes,specifically the species Aspergillus terreus, Aspergillus niger,Aspergillus niger var. awamori, Aspergillus ficuum, T. lanuginosus (alsoknown as H. lanuginosa), Myceliophthora thermophila, Peniophora lycii,Agrocybe pediades, Manascus anka, Paxillus involtus, or Trametespubescens. Suitable fungal phytases are described in Yamada et al.,1986, Agric. Biol. Chem. 322:1275-1282; Piddington et al., 1993, Gene133:55-62, EP 684,313; EP 0 420 358; EP 0 684 313; WO 98/28408; WO98/28409, JP 7-67635; WO 98/44125; WO 97/38096; WO 98/13480.

Modified phytases or phytase variants are obtainable by methods known inthe art, in particular by the methods disclosed in EP 897010; EP 897985;WO 99/49022; WO 99/48330.

Microorganism Used for Fermentation

Preferably microorganisms are used for the fermentation in step (d). Themicroorganism may be a fungal organism, such as yeast, or bacteria.Suitable bacteria may, e.g., be Zymomonas species, such as Zymomonasmobilis and E. coli. Examples of filamentous fungi include strains ofPenicillium species. Preferred organisms for ethanol production areyeasts, such as, e.g., Pichia or Saccharomyces. Preferred yeastaccording to the invention is Saccharomyces species, in particularSaccharomyces cerevisiae or bakers yeast.

Use of the Products Produced by the Method of the Invention

The ethanol obtained by the process of the invention may be used as,e.g., fuel ethanol; drinking ethanol, ie., potable neutral spirits, orindustrial ethanol, including fuel additive.

The invention in further aspect relates to use of a thermostable acidalpha-amylase or a thermostable maltogenic acid alpha-amylase in thesecondary liquefaction step in a process for production of ethanol;included is use of a thermostable acid alpha-amylase or a thermostablemaltogenic acid alpha-amylase in the secondary liquefaction step in theprocesses of the invention disclosed herein.

ADVANTAGES OF THE PROCESS OF THE INVENTION

By employing the thermostable acid alpha amylase of the invention in thesecondary liquefaction step, the process of the invention provides animproved process of producing ethanol. By the process of the inventionthe overall yield and/or process economy is increased. The process ofthe invention may make possible a lowering of the fermentation time.Further, the process of the invention may enhance the fermentationefficiency, e.g, by reducing the residual starch otherwise left over inthe fermentation. Furthermore, the process of the invention may reduceor eliminate the need for a pre-saccharification step.

By employing the thermostable maltogenic acid alpha amylase of theinvention in the secondary liquefaction step, the process of theinvention provides an improved process of producing ethanol. By theprocess of the invention the overall yield and/or process economy isincreased. The described thermostable maltogenic acid alpha-amylasewill, when used in the secondary liquefaction, produce a higher numberof fermentable sugars (maltose) as compared to the non-maltogenicalpha-amylases presently employed. This reduces the fermentation timeand/or the dosage of glucoamylase enzyme which is required to formfermentable sugars. Also as molecules of a lower molecular weight areformed the viscosity will be reduced as compared to non-maltogenicalpha-amylases. Reduced viscosity is desired in e.g., heat exchangersand dryers. Furthermore, the thermostable maltogenic acid alpha-amylaseby being active during fermentation conditions, and since this enzymehas an endo-breakdown mechanism it will in combination with theglucoamylase which is an exo-enzyme enable a more efficient hydrolysisof the starch during fermentation. Thus the process of the invention maymake possible a lowering of the fermentation time. The process of theinvention may enhance the fermentation efficiency, e.g., by reducing theresidual starch otherwise left over in the fermentation. Furthermore,the process of the invention may reduce or eliminate the need for apre-saccharification step.

Material & Methods

Determination of Viscosity

The mash is heated to a temperature of 50-70° C., depending on thetreatment. Following treatment viscosity is measured using a Haake VT02rotation based viscosimeter. The unit of viscosity is centipois (cps),which is proportionally related to the viscosity level.

Determination of Alpha-Amylase Activity (KNU)

The KNU is used to measure bacterial alpha-amylases with a high pHoptima.

Phadebas Assay

Alpha-amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Pharmacia Diagnostic) contain a cross-linked insoluble blue-coloredstarch polymer, which has been mixed with bovine serum albumin and abuffer substance and tabletted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin x ml of 60 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue color will be produced. The colorintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

Alternative Method

Alpha-amylase activity is determined by a method employing the PNP-G7substrate. PNP-G7 which is an abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide whichcan be cleaved by an endo-amylase. Following the cleavage, thealpha-glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow color and thus can be measured byvisible spectophometry at lambda=405 nm (400-420 nm). Kits containingPNP-G7 substrate and alpha-glucosidase is manufactured byBoehringer-Mannheim (cat. No. 1054635).

To prepare the substrate one bottle of substrate (BM 1442309) is addedto 5 ml buffer (BM1442309). To prepare the alpha-glucosidase one bottleof alpha-glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309).The working solution is made by mixing 5 ml alpha-glucosidase solutionwith 0.5 ml substrate.

The assay is performed by transforming 20 micro I enzyme solution to a96 well microtitre plate and incubating at 25° C., 200 micro I workingsolution, 25° C. is added. The solution is mixed and pre-incubated 1minute and absorption is measured every 15 sec. over 3 minutes at OD 405nm.

The slope of the time dependent absorption-curve is directlyproportional to the specific activity (activity per mg enzyme) of thealpha-amylase in question under the given set of conditions.

Determination of FAU Activity

One Fungal Alpha-Amylase Unit (FAU) is defined as the amount of enzyme,which breaks down 5.26 g starch (Merck Amylum solubile Erg. B.6, Batch9947275) per hour based upon the following standard conditions:

-   Substrate Soluble starch-   Temperature 37° C.-   pH 4.7-   Reaction time 7-20 minutes    Determination of Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity is measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard.

The standard used is AMG 300 L (from Novozymes A/S, glucoamylasewildtype Aspergillus niger G1, also disclosed in Boel et al., 1984, EMBOJ. 3 (5): 1097-1102) and WO 92/00381). The neutral alpha-amylase in thisAMG falls after storage at room temperature for 3 weeks from approx. 1FAU/mL to below 0.05 FAU/mL.

The acid alpha-amylase activity in this AMG standard is determined inaccordance with the following description. In this method, 1 AFAU isdefined as the amount of enzyme, which degrades 5.260 mg starch drymatter per hour under standard conditions.

Iodine forms a blue complex with starch but not with its degradationproducts. The intensity of color is therefore directly proportional tothe concentration of starch. Amylase activity is determined usingreverse colorimetry as a reduction in the concentration of starch underspecified analytic conditions.

Standard Conditions/Reaction Conditions: (Per Minute)

-   Substrate, Starch, approx. 0.17 g/L-   Buffer: Citate, approx. 0.03 M-   Iodine (I₂): 0.03 g/L-   CaCl₂: 1.85 mm-   pH: 2.50±0.05-   Incubation temperature: 40° C.-   Reaction time: 23 seconds-   Wavelength: lambda=590 nm-   Enzyme concentration: 0.025 AFAU/mL-   Enzyme working range, 0.01-0.04 AFAU/mL

If further details are preferred these can be found in EB-SM-0259.02/01available on request from Novozymes A/S, and incorporated by reference

EXAMPLES Example 1

Secondary Liquefaction Using a Thermostable Acidic Alpha Amylase

400 mL of a ground corn slurry was liquefied by a bacterialalpha-amylase and jet cooked at 105° C. for 5 min the resulting cornmash had 30% dry substance, DE 7 and pH 5.0. The mash was heated to 80°C. and the viscosity was measured to 500 CPS using a VT 180viscosimeter.

The mash was treated with a thermostable acidic alpha amylase fromAspergillus niger. The enzyme loading was 0.25 AFAU/g of dry matter,with 1 AFAU defined as the amount of enzyme that under standardconditions (37° C., pH 2.5 in 0.01 M acetate buffer) hydrolyzes 5.25 gstarch so that the hydrolyzed starch is only slightly colored byaddition of iodine-potassium-iodide.

After 30 min the viscosity and DE value were measured to 350 CPS and DE12, which shows that a final liquefaction of the corn mash was obtained.

Example 2

Secondary Liquefaction Using a Thermostable Maltogenic Acidic AlphaAmylase

400 mL of a ground corn slurry was liquefied by alpha-amylase TTC andjet cooked at 105° C. for 5 min; the resulting corn mash had 30% drysubstance, DE 7 and pH 5.0. The mash was heated to 80° C. and theviscosity was measured to 500 CPS using a VT 180 viscosimeter.

The mash was treated with a thermostable maltogenic acidic alpha amylasefrom Aspergillus niger having the amino acid sequence disclosed in SEQID NO: 1. The enzyme loading was 0.25 AFAU/g of dry matter, with 1 AFAUdefined as the amount of enzyme which under standard conditions (37° C.,pH 2.5 in 0.01 M acetate buffer) hydrolyzes 5.25 g starch so that thehydrolyzed starch is only slightly colored by addition ofiodine-potassium-iodide.

After 30 min the viscosity and DE value were measured to 350 CPS and DE12, which shows that a final liquefaction of the corn mash was obtained.

1-58. (canceled)
 59. A method of producing ethanol, comprising; a)liquefaction of a starch containing material in the presence of analpha-amylase; b) jet cooking the liquefied starch, c) a secondaryliquefaction in the presence of a thermostable acid alpha-amylase;wherein the thermostable acid alpha-amylase maintains more than 90% ofits activity for 1 hour at 70° C. using a DE 12 alpha-amylase TTCliquefied corn starch at 30% dry substance as substrate, pH 5.5, 0.1 Mcitrate buffer and 4.3 mM Ca+²⁺; d) saccharification; and e)fermentation to produce ethanol; wherein steps (a), (b), (c), and (d)are performed in the order (a), (b), (c), (d), and wherein step (e) isperformed simultaneously to or following step (d).
 60. The method ofclaim 59, further comprising recovering the ethanol.
 61. The method ofclaim 59, further comprising a pre-saccharification step which isperformed after the secondary liquefaction in step (c) and before step(d), wherein the pre-saccharification comprises treating the starch witha glucoamylase at a temperature in the range of 30-65° C.
 62. The methodof claim 59, wherein the starch containing material is selected from thegroup consisting of tubers, roots, whole grain, and any combinationthereof.
 63. The method of claim 59, wherein the starch containingmaterial is obtained from cereal.
 64. The method of claim 59, whereinthe starch containing material is selected from the group consisting ofcorns, cobs, wheat, barley, rye, milo, potatoes, and any combinationthereof.
 65. The method of claim 59, wherein the starch containingmaterial is whole grain selected from the group consisting of corn,wheat, barley, and any combination thereof.
 66. The method of claim 59,wherein the starch containing material is whole grain and said methodcomprises a step of milling the whole grain before step (a).
 67. Themethod of claim 59, wherein the starch containing material is obtainedby a process comprising milling of whole grain.
 68. The method of claim59, further comprising prior to step (a) the steps of: i) milling ofwhole grain; ii) forming a slurry comprising the milled grain and waterto obtain the starch containing material.
 69. The method of claim 68,wherein the milling is a dry milling step.
 70. The method of claim 68,wherein the milling is a wet milling step.
 71. The method of claim 59,wherein the starch-containing material is a side stream from starchprocessing.
 72. The method of claim 59, further comprising: (f)distillation to obtain the ethanol; wherein the fermentation in step (e)and the distillation in step (f) are carried out simultaneously orseparately/sequential.
 73. The method of claim 72; wherein the starchcontaining material is milled whole grain, said method furthercomprising the steps of: (g) separation of whole stillage produced bythe distillation in step (f) into wet grain and thin stillage; and (h)recycling the thin stillage to the starch containing material prior tostep (a).
 74. The method of claim 59, wherein the fermentation in step(e) is performed using a yeast.
 75. The method of claim 59, wherein thefermentation is carried out in the presence of glucoamylase, phytaseand/or protease.
 76. The method of claim 75, wherein the protease is anacid protease, a neutral protease or an alkaline protease.
 77. A methodfor producing ethanol, comprising: a) liquefaction of a starchcontaining material in the presence of an alpha-amylase; b) jet cookingthe liquefied starch; c) a secondary liquefaction in the presence of athermostable maltogenic acid alpha-amylase; wherein the thermostablemaltogenic acid alpha-amylase maintains more than 90% of its activityfor 1 hour at 70° C. using a DE 12 alpha-amylase TTC liquefied cornstarch at 30% dry substance as substrate, pH 5.5, 0.1 M citrate bufferand 4.3 mM Ca+²; d) saccharifaction; and e) fermentation to produceethanol; wherein steps (a), (b), (c) and (d) are performed in the order(a), (b), (c), (d) and wherein step (e) is performed simultaneously toor following step (d).
 78. The method of claim 77, wherein thethermostable maltogenic acid alpha-amylase having an amino acid sequencewhich is at least 95% identical to SEQ ID NO.
 1. 79. The method of claim77, wherein the thermostable maltogenic acid alpha-amylase is SEQ IDNO:
 1. 80. The method of claim 77, further comprising recovering theethanol.
 81. The method of claim 77, further comprising apre-saccharification step which is performed after the secondaryliquefaction in step (c) and before step (d), wherein thepre-saccharification comprises treating the starch with a glucoamylaseat a temperature in the range of 30-65° C.
 82. The method of claim 77,wherein the starch containing material is selected from the groupconsisting of tubers, roots, whole grain, and any combination thereof.83. The method of claim 77, wherein the starch containing material isobtained from cereal.
 84. The method of claim 77, wherein the starchcontaining material is selected from the group consisting of corns,cobs, wheat, barley, rye, milo, potatoes and any combination thereof.85. The method of claim 77, wherein the starch containing material iswhole grain selected from the group consisting of corn, wheat, barley,and any combination thereof.
 86. The method of claim 77, wherein thestarch containing material is whole grain and said method comprises astep of milling the whole grain before step (a).
 87. The method of claim77, wherein the starch containing material is obtained by a processcomprising milling of whole grain.
 88. The method of claim 77, furthercomprising prior to step (a) the steps of: i) milling of whole grain;ii) forming a slurry comprising the milled grain and water to obtain thestarch containing material.
 89. The method of claim 88, wherein themilling is a dry milling step.
 90. The method of claim 88, wherein themilling is a wet milling step.
 91. The method of claim 77, wherein thestarch-containing material is a side stream from starch processing. 92.The method of claim 77, further comprising: (f) distillation to obtainthe ethanol: wherein the fermentation in step (e) and the distillationin step (f) are carried out simultaneously or separately/sequential. 93.The method of claim 92; wherein the starch containing material is milledwhole grain, said method further comprising the steps of: (g) separationof whole stillage produced by the distillation in step (f) into wetgrain and thin storage; and (h) recycling the thin stillage to thestarch containing material prior to step (a).
 94. The method of claim77, wherein the fermentation in step (e) is performed using a yeast. 95.The method of claim 77, wherein the fermentation is carried out in thepresence of glucoamylase, phytase and/or protease.
 96. The method ofclaim 95, wherein the protease is an acid protease, a neutral proteaseor an alkaline protease.